Abstract:

Microplastics (MPs) are emerging global pollutants that have been identified as unique vectors that facilitate the dissemination of antibiotic resistance genes (ARGs) in aquatic environments. With the widespread adoption of biodegradable plastics as alternatives to conventional plastics, biodegradable MPs (BMPs) as their aging products have become ubiquitous in aquatic environments. Compared to conventional MPs, BMPs exhibit significantly enhanced surface adsorption properties, promoting the preferential enrichment of ARGs on their surfaces. However, the enrichment characteristics of ARGs in BMPs biofilms and the underlying mechanisms remain poorly understood. Furthermore, the health risks posed by ARGs enriched in BMPs biofilms have not been quantitatively assessed. This study investigated the distinct enrichment patterns and biological drivers of ARGs, identified the pathogenic hosts of ARGs, and evaluated the associated health risks of ARGs in biofilms formed on different types of BMPs. Three representative BMPs, polylactic acid (PLA), polyhydroxyalkanoate (PHA), and poly (butyleneadipate-co-terephthalate) (PBAT), were selected as target substrates for biofilm growth. Biofilm incubation experiments were conducted using natural river water as the culture medium in a custom mesocosm system. The surface properties of the three BMPs were characterized using Fourier transform infrared spectroscopy (FT-IR), and biofilm morphology and biomass were analyzed using scanning electron microscopy (SEM) and crystal violet assays, respectively. Metagenomic sequencing data were processed using bioinformatics approaches to profile ARGs, pathogenic hosts, microbial metabolic functions, and gene expression levels in BMPs biofilms. The health risks of ARGs in BMPs were quantified using an established ARGs health risk assessment framework. SEM images revealed visible biofilm formation on all three BMPs surfaces after 28 days of incubation. Biofilm biomass followed the order of PLA> PHA> PBAT, correlating with their varying surface hydrophilicities. The stronger hydrophilicity of the PLA surface likely enhanced the colonization of planktonic microorganisms in the surrounding water. Moreover, PLA biofilms exhibited the highest extracellular polymeric substance (EPS) content among the three types of BMPs, indicating a consistency between EPS and biomass variation in BMPs in freshwater ecosystems. FT-IR analysis revealed higher absorbance intensities corresponding to oxygen-containing functional groups on PLA, indicating that the PLA surface contained more oxygen-containing functional groups. The increase in the number of oxygen-containing functional groups in PLA significantly enhanced its surface adsorption capacity, facilitating microbial adhesion and biofilm formation. Principal coordinate analysis showed that the ARGs in all BMPs biofilm samples clustered together and were significantly separated from the ARGs in the surrounding water samples (r=0.985, p=0.001), suggesting a distinct distribution pattern of ARGs in BMPs biofilms compared to that in surrounding water. Multidrug and macrolide-lincosamide-streptogramin resistance genes were the most abundant ARGs in all samples. The relative abundance of ARGs in BMPs biofilms (3.21×10⁴-3.74×10⁴ TPM) was significantly higher than that in the surrounding water (1.63×10⁴ TPM), with PLA, PHA, and PBAT biofilms exhibiting 2.29-fold, 2.02-fold, and 1.96-fold higher ARGs abundance, respectively. The highest ARGs enrichment on PLA biofilms was attributed to the elevated biofilm biomass and EPS production, likely due to the abundant oxygen-containing functional groups on its surface. Additionally, PLA degradation may create additional microbial niches, further promoting ARGs enrichment. Linear Discriminant Effect Size analysis showed that BMPs could selectively enrich ARGs, with significant variations in ARGs enrichment patterns among different types of BMPs. Notably, PLA not only exhibited a stronger ARGs enrichment ability but also preferentially enriched ARGs carried by pathogens. Metagenomic binning analysis identified 33 pathogenic metagenome-assembled genomes (MAGs) harboring 76 ARGs subtypes, primarily multidrug (27 subtypes), peptide (7 subtypes), and phenicol (7 subtypes) resistance genes. Notably, 30, 32, and 31 pathogenic MAGs were detected in the PLA, PHA, and PBAT biofilms, respectively, significantly exceeding those in the surrounding water (three MAGs). The relative abundances of pathogenic MAGs reached 4.41×10⁴ TPM (PLA), 9.25×10⁴ TPM (PHA), and 3.71×10⁵ TPM (PBAT), which were substantially higher than those in the surrounding water (3.91×10⁴ TPM). These findings demonstrate that BMPs significantly increase the diversity and relative abundance of ARGs pathogenic hosts in aquatic environments. Furthermore, Hydrogenophaga emerged as the dominant ARGs pathogenic host in both BMPs biofilms and the surrounding water, but its relative abundance was markedly higher in BMPs biofilms. Metabolic function and gene expression analyses revealed that high metabolic activity, including two-component systems, ABC transporters, and quorum sensing, along with upregulated key genes (e.g., oxidative stress genes osmY and osmC and global regulators korA and korB), drove ARGs enrichment in BMPs biofilms. Notably, the high proportions of two-component systems, ABC transporters, and quorum sensing in PLA biofilms were 1.08-1.82, 1.16-1.88, and 1.05-1.77 times higher, respectively, than those in PHA biofilms, PBAT biofilms, and surrounding water, which stimulated the proliferation of ARGs. Consequently, PLA biofilms exhibited the highest relative abundance of ARGs among the three BMPs types. These results suggest that reducing metabolic activity and related key gene expression could mitigate ARGs enrichment in BMPs biofilms. Health risk assessment indicated that ARGs risks in BMPs biofilms were significantly higher than in surrounding water, with PLA, PHA, and PBAT biofilms showing 4.27-fold, 3.28-fold, and 3.24-fold higher ARGs risk values, respectively. The high health risk of ARGs in BMPs biofilms poses a potential threat to aquatic ecological environments and may exert profound effects on human health through transmission via the food chain. BMPs serve as vectors for ARGs, BMPs provide stable habitats for antibiotic-resistant bacteria, and facilitating the spread of ARGs. In aquatic environments, BMPs attached to ARGs are readily ingested by primary consumers and subsequently transferred through the food chain to higher organisms, including humans. As crucial nodes in the food web, these aquatic organisms may accumulate antibiotic-resistant bacteria and ARGs, ultimately introducing them into humans through the consumption of aquatic foods. The highest ARGs risk in PLA biofilms was primarily due to the enrichment of multidrug resistance genes. The accumulation of ARGs on BMPs surfaces threatens aquatic ecosystems and may propagate through the food chain, ultimately endangering human health. Overall, this study provides a comprehensive evaluation of the occurrence patterns of ARGs and their corresponding biological drivers in BMPs biofilms within freshwater ecosystems. The findings offer valuable reference data for understanding the dissemination patterns of antimicrobial resistance in BMPs and establish a scientific foundation for developing effective strategies to mitigate microplastic pollution in aquatic environments.

Key words: biodegradable microplastics, biofilms, antibiotic resistance genes, pathogenic hosts, health risk

摘要：

可生物降解微塑料（Biodegradable microplastics，BMPs）已被证实是促进水生环境中抗生素抗性基因（Antibiotic resistance genes，ARGs）传播的重要载体，加剧了环境健康风险。然而，不同类型BMPs生物膜上富集的ARGs健康风险的定量评估尚不明晰。通过构建中宇宙实验体系，结合宏基因组学分析，探究了聚乳酸（Polylactic acid，PLA）、聚羟基脂肪酸酯（Polyhydroxyalkanoates，PHA）和聚己二酸对苯二甲酸丁二醇酯（Poly (butyleneadipate-co-terephthalate)，PBAT）3种BMPs生物膜上ARGs及其病原宿主的富集特征和生物驱动因素，并定量评估相关健康风险。结果表明，经28 d孵育后3种BMPs表面均形成明显的生物膜，且PLA生物膜的生物量和胞外聚合物含量最高。BMPs生物膜上ARGs的相对丰度是周围水体的1.97-2.29倍，PLA生物膜上ARGs的相对丰度最高（3.91×10⁴ TPM）。氢噬胞菌属（Hydrogenophaga）的细菌成员是所有BMPs生物膜上ARGs的主要病原宿主。代谢功能和关键基因分析表明，PLA生物膜上的微生物通过调控双组分系统、ABC转运蛋白和群体感应促进了ARGs的富集。健康风险评估显示，BMPs生物膜上ARGs健康风险是周围水体的3.24-4.27倍，且PLA生物膜上ARGs的健康风险最高。研究结果为理解BMPs介导的抗生素耐药性传播提供了重要参考数据，同时为制定水环境微塑料污染防控策略奠定了科学基础。

关键词: 可生物降解微塑料, 生物膜, 抗生素耐药基因, 病原宿主, 健康风险